Thiosemicarbazones Synthesized from Acetophenones: Tautomerism, Spectrometric Data, Reactivity and Theoretical Calculations

Tautomeric forms of Thiosemicarbazones have been investigated by spectrometric methods, their chemical reactivity and theoretical calculations of the relative tautomers stabilities. The mass spectral fragmentation of thiosemicarbazones synthesized from acetophenones has been studied by CG/MS. The analysis of the corresponding spectra shows not only the regular fragmentation mechanisms but homolytic ruptures from even-electron species. H NMR spectra exhibit signals for the most intense open thioketo tautomeric structure, although when using TFA a ring structure is observed in the corresponding tautomeric equilibrium. Density Functional Theory calculations (DFT) also provide evidence to support the experimental observations by GC-MS and H NMR. Methylation reactions give support to the occurrence of the thioenol tautomeric form which would be the second most abundant according to the Density Functional Theoretical calculations.

These substances exhibit a wide variety of biological activities as, for example, antitumor, antibacterial, antiviral, antiprotozoal activity and cytotoxic effects [1].
Chagas-Mazza disease is a widely spread disease in rural areas of South and Central America and it was reported that these compounds may constitute an alternative treatment. Thiosemicarbazones can inhibit cruzain, which is the major cysteine protease present in Trypanosome cruzi that is expressed in every step of the life of the parasite that causes the mentioned disease [2].
These molecules exhibit tautomeric equilibria what is certainly relevant because the occurrence of a particular tautomer determines the reactivity. Thiosemicarbazones can exhibit several tautomers [3] with open and ring structures.
Open keto-forms are certainly the most stable ones. Scheme 1 shows all the possible tautomeric forms for the thiosemicarbazone of acetophenone. Ring structures have been investigated by NMR [4] and it has been shown that the solvent polarity modifies the equilibria. The 4-methyl and the 2-methyl-thiosemicarbazones of acetone convert readily into the corresponding 1,2,4-triazolidine-3-thiones in deuterated trifluoroacetic acid, while they are present exclusively as the open-chain forms in d 6 -dimethylsulfoxide.
Semicarbazones and thiosemicarbazones can form complexes with metals and several of them have exhibit biological activity (particularly antimicrobial and antitumor activity have been reported) [5] [6] [7]. Additionally, they have been used as corrosion inhibitors of certain alloys [8].
In this work, a study of thiosemicarbazones synthetized from substituted acetophenones is carried out by means of gas chromatography-mass spectrometry, 1 H-NMR and theoretical calculations. It has been reported that the GCMS spectra revealed not only the dissociation of the thiosemicarbazones in the injection port but also the formation of compounds of higher molecular weight with longer retention times [9]. Additionally, unusual fragmentation routes (homolytic ruptures of even-electron ions) are observed. Density Functional Theory (DFT) [10] [11] [12] [13] calculations were carried out to investigate stability among tautomeric forms in order to have answers on the reactivity and spectral behavior. It is well known that computational tools are of great aid to gain insights that could help to understand and predict the structure, stability, and reactivity of organic compounds. Among them, methods based on the DFT have proven to be very effective.

Materials and Methodology
The compounds under study were synthesized by adapting literature procedures [14] [15]. Table 1

Reagents
All the reagents have been commercially acquired and have been used without further treatment: in the interface, 280˚C in the ion source and the oven ramp started at 100˚C (5 min) and ended at 350˚C with a heat rate of 10˚C·min −1 . The electron energy was 70 eV and the pressure in the mass spectrometer was about 10 −5 torr, thus precluding ion-molecule reactions. These determinations were carried out to examine the mass spectrometric behavior of these compounds which could exhibit tautomeric equilibria and are thermolabile.  Table   2). Deuterium from the solvent was used as the lock and TMS as the internal standard. Sample concentration was 20 mg/ml. Measurements were performed at 25˚C.

Computational Procedures
Geometry optimization of the molecules involved in the processes was accomplished using the Becke's three parameters hybrid density functional [16] with the gradient-corrected correlation functional due to Lee, Yang and Parr [17], a combination that gives rise to the well-known B3LYP functional [18]. In order to confirm that the obtained structures are local minimum on the potential energy surface, the eigenvalues of the hessian matrix of the electronic energy, with respect to the atomic coordinates, were inspected. The double-hybrid RI-PWPB95 functional [19], including the D3 dispersion correction due to Grimme [20] and the Becke-Johnson damping function [21], were used to obtain a better estimation of total electronic energies from single point calculations on previously optimized geometries. The def2-SVP and the def2-QZVPP basis sets [22] were utilized for geometry optimizations and for single-point calculations, respectively. All the calculations were carried out with the ORCA package [23].  Figure 1 shows the total ion chromatogram (TIC) after injection of the thiosemicarbazone of 4-methylacetophenone (IV), which has been object of study in a previous work [9].

Gas Chromatography-Mass Spectrometry
The mass spectrum of the peak at 5.2 minutes due to the thiosemicarbazone of acetophenone (I) is included in Table 1. As it can be seen, there are two chromatographic peaks. Peak purity allowed discarding co-elution in any of the observed peaks. The second peak at 10.2 minutes is due to a compound of higher molecular weight that is formed in the injection port [9].  For all studied thiosemicarbazones the easy loss of ammonia is also the reason for the molecular ion absence in their spectra.
The ion at m/z 168 in the spectrum of the thiosemicarbazone of 4-chloroacetophenone (II) is due to an easy loss of S=C=NH from the molecular ion.
The mass spectrum of the thiosemicarbazone of 4-nitroacetophenone (III), has the base peak at m/z 117 (not included in Table 1) which is the loss of a nitro-group, typical of nitroaromatics, from the ion at m/z 163 rendering the radical ion C 6 H 4 CH 3 CN + . Its structure would be similar to that one for the m/z 118 in which also exhibits the same fragmentation, m/z 205, 31.5%. Theoretical calculations (see below) support the highest stability of the thioketo-form (T 1 ) followed by the thioenol-form (T 2 ). It is assumed that the methylation reaction takes place with the little abundant thiol-tautomer.

1 H NMR
The 1 H NMR spectrum for the thiosemicarbazone of 4-nitroacetophenone (III)   in dimethylsulfoxide-d 6 is described in Table 2. The registered spectrum would correspond to the open-chain thioketo-tautomer. The NH 2 group in all cases is responsible for two signals in the 1 H NMR spectra. The reason for this behavior could be assigned to a "slow" C-N rotation in the thioamide-moiety along with the interaction of one of the protons with the sp2 nitrogen atom establishing a five-member ring hydrogen bonding which would shift the corresponding signal to lower fields.
An interesting result is observed in the spectrum of III in deuterated trifluoroacetic acid (Figure 4)   The signal assignments in the spectra suggest that the ring structure might correspond to the tautomer-form T 9 . Additional support has been resourced by theoretical calculations.

Theoretical Calculations
Relative formation energies for the tautomers of thiosemicarbazones in the gas phase are depicted in Table 4. Some DFT calculations in ethanolic solutions have been reported [25]. Fifteen tautomers are possible taking into consideration  Among the open-chain forms, the next most stable is T 2 what is consistent with the methylation results and the corresponding mass spectrum (Figure 3).
The bond dissociation energy for the homolytic fragmentation of the N-N bond of the ion shown in Scheme 2 was calculated to be 3.42 eV (at 298˚K). For comparison purposes, the bond dissociation energy for the homolysis of an even-electron ion in which the nitrogen atom is bound to an sp3 carbon atom, was also calculated. In this case, the bond dissociation energy is considerably higher, namely 4.67 eV, rendering also the ion at m/z 119 (Scheme 5). So that, this ion would be mainly formed by the homolytic route. The analogous observation was reported for the homolytic rupture between sp-and sp2-nitrogens atoms (compared to the rupture between sp-nitrogen and sp2-carbon atoms) for related structures [9].
T 1 is the most stable open-chain tautomer (thioketo-), followed by T 2 (thioenol-). The cyclic structures are considered separately since their formation implies, at least, a cyclo-addition reaction. Among the ring structures, T 9 would be the most stable (cycloaddition of T 2 involving the SH group) followed by T 12 (with a thiol-group), with formation energy very closed to that one of T 13 (with a thiocarbonyl-group). Both T 9 and T 12 exhibit a 4-center conjugated system (-NH-N=C-NH 2 and -NH-N=C-SH respectively what might account for their relative stabilities). It is interesting that T 12 would be the cycloaddition product from T 2 and T 13 that one from T 1 , in both cases the cycloaddition would involve the NH 2 group (Scheme 6). As experimentally observed, high acidic media is necessary. Very good support for the occurrence of the tautomeric ring structure is provided by NMR studies in highly acidic media (Trifluoracetic acid).
This result is observed for all the thiosemicabazones under study.
Additionally, there is a clear indication of the stabilizing effect of the oxymethyl-group (compound V) on the formation energies (